RESUMEN

Listeria monocytogenes is a foodborne bacterial pathogen that causes listeriosis. Positive regulatory factor A (PrfA) is a pleiotropic master activator of virulence genes of L. monocytogenes that becomes active upon the entry of the bacterium into the cytosol of infected cells. L. monocytogenes can survive and multiply at low temperatures; this is accomplished through the maintenance of appropriate membrane fluidity via branched-chain fatty acid (BCFA) synthesis. Branched-chain α-keto acid dehydrogenase (BKD), which is composed of four polypeptides encoded by lpd, bkdA1, bkdA2, and bkdB, is known to play a vital role in BCFA biosynthesis. Here, we constructed BKD-deficient Listeria strains by in-frame deletion of lpd, bkdA1, bkdA2, and bkdB genes. To determine the role in in vivo and in vitro, mouse model challenges, plaque assay in murine L2 fibroblast, and intracellular replication in J744A.1 macrophage were conducted. BKD-deficient strains exhibited defects in BCFA composition, virulence, and PrfA-regulon function within the host cells. Transcriptomics analysis revealed that the transcript level of the PrfA-regulon was lower in ΔbkdA1 strain than those in the wild-type. This study demonstrates that L. monocytogenes strains lacking BKD complex components were defective in PrfA-regulon function, and full activation of wild-type prfA may not occur within host cells in the absence of BKD. Further study will investigate the consequences of BKD deletion on PrfA function through altering BCFA catabolism.IMPORTANCEListeria monocytogenes is the causative agent of listeriosis, a disease with a high mortality rate. In this study, we have shown that the deletion of BKD can impact the function of PrfA and the PrfA-regulon. The production of virulence proteins within host cells is necessary for L. monocytogenes to promote its intracellular survival and is likely dependent on membrane integrity. We thus report a link between L. monocytogenes membrane integrity and the function of PrfA. This knowledge will increase our understanding of L. monocytogenes pathogenesis, which may provide insight into the development of antimicrobial agents.

Asunto(s)

Proteínas Bacterianas , Listeria monocytogenes , Listeriosis , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Listeria monocytogenes/enzimología , Listeria monocytogenes/metabolismo , Ratones , Animales , Virulencia , Listeriosis/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Ácidos Grasos/biosíntesis , Ácidos Grasos/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/genética , Regulación Bacteriana de la Expresión Génica , Macrófagos/microbiología , Femenino , Línea Celular

RESUMEN

Chronic subclinical infection with the aetiological agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, presents challenges for the clinical management of disease in farmed salmonids and for prevalence estimation. Harvested salmon sampled at processing plants provide the opportunity to describe subclinical outcomes of BKD using gross necropsy observations and diagnostic test results in farmed Atlantic salmon (Salmo salar L.) populations that are apparently healthy (i.e. alive at harvest) but naturally exposed to R. salmoninarum infection. Sampling of farmed salmon (Population A, n = 124 and Population B, n = 160) was performed immediately post-slaughter as fish were being processed at a plant in New Brunswick, Canada. Populations were selected based on planned harvests from sites with histories of recent exposure events related to clinical BKD as evidenced by the site veterinarian's diagnosis of mortality attributable to BKD: One site (Pop A) had recently increasing mortalities attributed to BKD, and the other site (Pop B) had ongoing low-level mortalities with BKD pathology. As expected with the different exposure histories, Pop A had a higher percentage (57.2%) of R. salmoninarum culture-positive kidney samples compared with similar fish samples in Pop B (17.5%). Diagnosis of R. salmoninarum by gross granulomatous lesions in internal visceral organs, bacterial culture and identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using different swab transport methods, and molecular detection methods (quantitative PCR, qPCR) were compared. Agreement of culture-positive percentages at the sample level was moderate (kappa: 0.61-0.75) among specimens collected using different kidney sampling methods in Pop A and Pop B. The highest proportion of R. salmoninarum-positive cultures occurred when kidney tissues were transported to the laboratory and inoculated directly onto agar using a swab (94% of cultures from Pop A and 82% from Pop B when fish were positive by any culture method). Fish with cumulative lesion scores (severity of granulomatous lesions in 3 different visceral organs) of >4 were all culture positive, and when compared with non-lesioned fish, had substantially higher odds of being culture positive: Pop A: odds ratio (OR) = 73, 95% confidence interval (CI) (7.91, 680.8); Pop B: OR = 66, 95% CI (6.12, 720.7). Our study found that onsite postmortem examinations with severity scores of gross granulomatous lesions were predictive of positive culture results for R. salmoninarum, and they were a useful proxy for assessing prevalence in apparently healthy populations with subclinical infection.

Asunto(s)

Enfermedades de los Peces , Enfermedades Renales , Micrococcaceae , Salmo salar , Animales , Infecciones Asintomáticas , Enfermedades de los Peces/microbiología , Enfermedades Renales/epidemiología , Canadá , Pruebas Diagnósticas de Rutina

RESUMEN

Renibacterium salmoninarum (Rs) is the etiological agent of bacterial kidney disease (BKD), which significantly affects farmed and wild salmonids worldwide. Although the whole genome of Rs (~3.1 million nucleotides) is highly conserved, genomic epidemiology analyses have identified four sub-lineages from Chilean isolates. A total of 94 Rs genomes from the BIGSdb aquaculture database were aligned and compared using bioinformatics tools, identifying 2199 independent single-nucleotide polymorphisms (SNPs) spread along the genome. A detailed analysis of the distribution of the SNPs showed five local zones of a length in the range of 10-15 kbp that should be used to unambiguously identify a specific sub-lineage. Based on the Rs type strain DSM 20767T , we designed multiplex PCR primers that produce specific amplification products which were further sequenced by the Sanger method to obtain the genotype of the sub-lineage. For the genetic typing, we evaluated 27 Rs isolates recovered from BKD outbreaks from different fish species and regions of Chile. Based on the findings reported here, we propose the PCR approach as a valuable tool for the rapid and reliable studying of the relationships between Rs isolates and the different sub-lineages without requiring the sequencing of the entire genome.

Asunto(s)

Enfermedades de los Peces , Micrococcaceae , Animales , Salmón , Chile , Enfermedades de los Peces/microbiología , Acuicultura

RESUMEN

Renibacterium salmoninarum, a Gram-positive intracellular pathogen, is the causative agent of bacterial kidney disease (BKD), the impacts of which are high mortalities and economic losses for the salmon industry. This study provides novel analyses for the whole-genome sequences of 50 R. salmoninarum isolates and the reference strain ATCC 33209 using a pan-genomic approach to elucidate phylogenomic relationships and identify unique and shared genes associated with pathogenicity and infection mechanisms. Genome size varied from 3,061,638 to 3,155,332 bp; gene count from 3452 to 3580; and predicted coding sequences from 3402 to 3527. Comparative analyses revealed an open, but approaching closed, pan-genome. The pan-genome analysis recovered 4064 genes, with a core genome containing 3306 genes. Phylogenetic analysis of R. salmoninarum showed high genomic homogeneity, apart from one isolate obtained from Salmo trutta in Norway. All genomes presented the 57-kDa protein (p57). Strain ATCC 33209 and the Chilean isolates H-2 and DJ2R presented two copies of the msa gene, while the remaining isolates had one copy. The pan-genome analysis further identified differences in the number of copies and length of the signalling peptide for p57, the principal virulence factor reported for this bacterium. This heterogeneity could be associated with the secretion levels of p57, potentially influencing virulence. Additionally identified were numerous common genes related to iron uptake, the stress response and regulation, and cell signalling-all of which constitute the pathogenic repertoire of R. salmoninarum. This investigation provides information that is applicable in future studies for identifying therapeutic targets and/or for designing new strategies (e.g., vaccines) to prevent BKD infections in salmon farming.

Asunto(s)

Enfermedades de los Peces , Enfermedades Renales , Micrococcaceae , Animales , Enfermedades de los Peces/microbiología , Genómica , Enfermedades Renales/microbiología , Micrococcaceae/genética , Filogenia , Renibacterium , Salmón , Factores de Virulencia/genética , Factores de Virulencia/metabolismo

RESUMEN

Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum (Rs), can be transmitted both horizontally and vertically and there is no available cure or prophylaxis. The control of BKD requires continuous surveillance, which is challenging in aquaculture as well as in programs for conservation and restoration of salmonid fish strains. BKD is a notifiable disease in Sweden and is monitored through the mandatory health control program using a polyclonal ELISA for detection of the Rs p57 protein in kidney. Fish must be killed for sampling, an obvious disadvantage especially regarding valuable broodfish. The present study shows that gill-/cloacal swabs collected in vivo for real-time PCR (qPCRgc ), allow a sensitive and specific detection of Rs. The sensitivity of qPCRgc was estimated to 97.8% (credible interval (ci) 93.8%-100%) compared to 98.3% (ci 92.7%-100%) and 48.8% (ci 38.8%-58.8%) of kidney samples for qPCR (qPCRk ) and ELISA (ELISAk ) respectively, by use of the Bayesian Latent Class Analysis (BLCA). Since the goal of the program is eradication of BKD the most sensitive test is preferrable. Using qPCRgc instead of ELISAk will result in a lower false negative rate and can be useful for surveillance in aquaculture and in breeding programs with valuable fish. However, a higher false positive rate warrants confirmatory lethal testing before a previously Rs negative farm is subject to restrictions.

Asunto(s)

Infecciones Bacterianas , Enfermedades de los Peces , Enfermedades Renales , Micrococcaceae , Animales , Teorema de Bayes , Femenino , Enfermedades de los Peces/diagnóstico , Enfermedades de los Peces/microbiología , Riñón/microbiología , Enfermedades Renales/diagnóstico , Enfermedades Renales/microbiología , Enfermedades Renales/veterinaria , Masculino , Micrococcaceae/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Renibacterium

RESUMEN

Bacterial kidney disease (BKD) can be a devastating bacterial infection in salmonids, and it is present in aquaculture throughout the world. BKD is caused by the Gram-positive facultative intracellular bacterium Renibacterium salmoninarum (R. salmoninarum) that is spread both horizontally and vertically. Disease signs include external ulcerations and blisters and internal signs such as organ swelling, granulomas, petechiae and ascites. In Sweden, BKD accounts for a significant income loss in aquacultures due to expensive decontamination of the facility and increased disease susceptibility for the immunocompromised fish leading to higher mortality rates. In addition, uncontrolled spread in aquaculture may threaten the survival of wild fish populations. The aim of our study was to investigate the prevalence of R. salmoninarum in wild salmonids caught in Swedish waters where net pen farms with a recent history of BKD are present. Four rivers with at least one BKD-positive or recently BKD-positive farm were selected. In addition, we evaluated the use of environmental DNA (eDNA) for surveillance and monitoring of ongoing infections at these locations. In total, 1058 fish were sampled from four different river systems, and of them 52 (4.9%) were positive for R. salmoninarum by antigen ELISA. Surprisingly, these fish were not evenly distributed between the four river systems, but 50 were caught in the same river (Ljungan). This accounts for an alarmingly high rate of 17% R. salmoninarum-positive samples in wild salmonids in this area. This number is far above what was expected and clearly shows the risk with an open farming system as well as the importance of effective health monitoring programmes to avoid an uncontrolled spread of the disease. The use of eDNA for monitoring BKD is somewhat difficult to evaluate. Few of the water samples analysed were PCR positive for R. salmoninarum (2 of 38) and those were collected where no ELISA positive fish were identified. In addition to water, sediment samples were collected under a net pen farm that had recently slaughtered all fish due to ongoing R. salmoninarum infections. Sediment samples are more promising than water as 4 of 5 samples at one farming facility where positive for R. salmoninarum. Thus, sediment samples may be valuable for monitoring potential ongoing BKD in farms, without the need to sacrifice valuable fish.

Asunto(s)

Enfermedades de los Peces , Enfermedades Renales , Micrococcaceae , Salmonidae , Animales , Enfermedades de los Peces/microbiología , Enfermedades Renales/veterinaria , Micrococcaceae/genética , Renibacterium , Suecia/epidemiología

RESUMEN

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1ß, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

Asunto(s)

Enfermedades de los Peces/inmunología , Infecciones por Bacterias Grampositivas/inmunología , Enfermedades Renales/inmunología , Perciformes/microbiología , Inmunidad Adaptativa/genética , Animales , Carga Bacteriana , Técnicas Bacteriológicas , Enfermedad Crónica , Susceptibilidad a Enfermedades , Enfermedades de los Peces/microbiología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Ontología de Genes , Infecciones por Bacterias Grampositivas/genética , Infecciones por Bacterias Grampositivas/microbiología , Riñón Cefálico/inmunología , Riñón Cefálico/metabolismo , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunidad Celular/genética , Enfermedades Renales/genética , Enfermedades Renales/microbiología , Perciformes/genética , Perciformes/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Renibacterium , Especificidad de la Especie , Organismos Libres de Patógenos Específicos

RESUMEN

Surveillance of antibiotic resistance is of paramount importance for animal welfare and production. Despite aquaculture being a main source of animal protein, studies on antibiotic susceptibility in fish pathogens are scarce. Renibacterium salmoninarum, the aetiological agent of bacterial kidney disease (BKD), is one of the most common bacterial pathogens affecting salmon farming. In this work, we present an analysis of susceptibility patterns using determinations of minimum inhibitory concentration (MIC) for 65 field isolates, which were collected over seven years (2013-2019) from Atlantic salmon (Salmo salar) and coho salmon (Oncorhynchus kisutch) farms across southern Chile. The MIC protocol described by the Clinical Laboratory Standards Institute (CLSI) was used, but with microdilution instead of macrodilution and eight instead of four days of incubation. Two laboratories independently conducted analyses to provide data on the epidemiological cut-off values for R. salmoninarum to florfenicol, oxytetracycline and erythromycin. By using two calculation methods, our results provide evidence for an evolving subpopulation of non-wild-type isolates for the macrolide erythromycin, which is consistent with the respective treatment frequencies prescribed against BKD. Contrasting with what was expected, R. salmoninarum isolates were most susceptible to florfenicol and oxytetracycline, both of which are widely used antibiotics currently used in the Chilean salmon industry. The presented findings can serve as a reference for national or international antibiotic surveillance programmes, for both MIC interpretation and to identify emerging resistance to the conventional drugs used in BKD management. Finally, our results indicate that an 8-day incubation period for establishing MIC values of R. salmoninarum should be considered in a future revision of the CLSI guidelines.

Asunto(s)

Antibacterianos/farmacología , Enfermedades de los Peces/microbiología , Infecciones por Bacterias Grampositivas/veterinaria , Pruebas de Sensibilidad Microbiana/veterinaria , Animales , Acuicultura , Chile , Enfermedades de los Peces/tratamiento farmacológico , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana/métodos , Oncorhynchus kisutch , Renibacterium/efectos de los fármacos , Salmo salar

RESUMEN

Bacterial Kidney Disease (BKD), which is caused by a Gram-positive, intracellular bacterial pathogen (Renibacterium salmoninarum), affects salmonids including Atlantic salmon (Salmo salar). However, the transcriptome response of Atlantic salmon to BKD remained unknown before the current study. We used a 44K salmonid microarray platform to characterise the global gene expression response of Atlantic salmon to BKD. Fish (~54 g) were injected with a dose of R. salmoninarum (H-2 strain, 2 × 108 CFU per fish) or sterile medium (control), and then head kidney samples were collected at 13 days post-infection/injection (dpi). Firstly, infection levels of individuals were determined through quantifying the R. salmoninarum level by RNA-based TaqMan qPCR assays. Thereafter, based on the qPCR results for infection level, fish (n = 5) that showed no (control), higher (H-BKD), or lower (L-BKD) infection level at 13 dpi were subjected to microarray analyses. We identified 6,766 and 7,729 differentially expressed probes in the H-BKD and L-BKD groups, respectively. There were 357 probes responsive to the infection level (H-BKD vs. L-BKD). Several adaptive and innate immune processes were dysregulated in R. salmoninarum-infected Atlantic salmon. Adaptive immune pathways associated with lymphocyte differentiation and activation (e.g., lymphocyte chemotaxis, T-cell activation, and immunoglobulin secretion), as well as antigen-presenting cell functions, were shown to be differentially regulated in response to BKD. The infection level-responsive transcripts were related to several mechanisms such as the JAK-STAT signalling pathway, B-cell differentiation and interleukin-1 responses. Sixty-five microarray-identified transcripts were subjected to qPCR validation, and they showed the same fold-change direction as microarray results. The qPCR-validated transcripts studied herein play putative roles in various immune processes including pathogen recognition (e.g., tlr5), antibacterial activity (e.g., hamp and camp), regulation of immune responses (e.g., tnfrsf11b and socs1), T-/B-cell differentiation (e.g., ccl4, irf1 and ccr5), T-cell functions (e.g., rnf144a, il13ra1b and tnfrsf6b), and antigen-presenting cell functions (e.g., fcgr1). The present study revealed diverse immune mechanisms dysregulated by R. salmoninarum in Atlantic salmon, and enhanced the current understanding of Atlantic salmon response to BKD. The identified biomarker genes can be used for future studies on improving the resistance of Atlantic salmon to BKD.

Asunto(s)

Inmunidad Adaptativa/genética , Enfermedades de los Peces/genética , Enfermedades de los Peces/microbiología , Perfilación de la Expresión Génica , Inmunidad Innata/genética , Salmo salar/genética , Salmo salar/microbiología , Transcriptoma , Animales , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Renibacterium , Reproducibilidad de los Resultados , Transducción de Señal

RESUMEN

Post-translational modifications of proteins enable swift physiological adaptation of cells to altered growth conditions and stress. Aside from protein phosphorylation, acetylation on ε-amino groups of lysine residues (N-ε-lysine acetylation) represents another important post-translational modification of proteins. For many bacterial pathogens, including the whooping cough agent Bordetella pertussis, the role and extent of protein acetylation remain to be defined. We expressed in Escherichia coli the BP0960 and BP3063 genes encoding two putative deacetylases of B. pertussis and show that BP0960 encodes a lysine deacetylase enzyme, named Bkd1, that regulates acetylation of a range of B. pertussis proteins. Comparison of the proteome and acetylome of a Δbkd1 mutant with the proteome and acetylome of wild-type B. pertussis (PRIDE ID. PXD016384) revealed that acetylation on lysine residues may modulate activities or stabilities of proteins involved in bacterial metabolism and histone-like proteins. However, increased acetylation of the BvgA response regulator protein of the B. pertussis master virulence-regulating BvgAS two-component system affected neither the total levels of produced BvgA nor its phosphorylation status. Indeed, the Δbkd1 mutant was not impaired in the production of key virulence factors and its survival within human macrophages in vitro was not affected. The Δbkd1 mutant exhibited an increased growth rate under carbon source-limiting conditions and its virulence in the in vivo mouse lung infection model was somewhat affected. These results indicate that the lysine deacetylase Bkd1 and N-ε-lysine acetylation primarily modulate the general metabolism rather than the virulence of B. pertussis.

Asunto(s)

Proteínas Bacterianas , Lisina , Acetilación , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bordetella pertussis/genética , Regulación Bacteriana de la Expresión Génica , Lisina/metabolismo , Ratones , Virulencia

RESUMEN

Bacterial kidney disease (BKD) is widespread in many areas of the world and can cause substantial economic losses for the salmon aquaculture industry. The objective of this study was to investigate the pathophysiological response and gene expression profiles related to the immune response at different water temperatures and to identify the best immunopathological biomarkers to define a phenotype of resistance to BKD. The abundance of msa transcripts of R. salmoninarum in the head kidney was significantly higher in infected fish at 11°C. R. salmoninarum induced significantly more severe kidney lesions, anemia and impaired renal function at 11°C. In addition, the expression pattern of the genes related to humoral and cell-mediated immune responses in infected fish at 11 and 15°C was very similar, although R. salmoninarum induced a significantly greater downregulation of the adaptive immune response genes at the lower water temperature. These results could be due to a suppressed host response directly related to the lowest water temperature and/or associated with a delayed host response related to the lowest water temperature. Although no significant differences in survival rate were observed, fish infected at the lowest temperature showed a higher probability of death and delayed the mortality curve during the late stage of infection (35 days after infection). Thirty-three immunopathological biomarkers were identified for potential use in the search for a resistance phenotype for BKD, and eight were genes related specifically to the adaptive cell-mediated immune response.

Asunto(s)

Enfermedades de los Peces/inmunología , Enfermedades de los Peces/microbiología , Infecciones por Bacterias Grampositivas/veterinaria , Salmo salar/inmunología , Salmo salar/microbiología , Animales , Frío , Resistencia a la Enfermedad/genética , Ambiente , Infecciones por Bacterias Grampositivas/inmunología , Inmunidad Celular/genética , Inmunidad Celular/inmunología , Renibacterium , Salmo salar/genética , Transcriptoma , Agua

RESUMEN

Bacterial Kidney Disease (BKD) is an economically significant disease in salmonid aquaculture and commonly requires antibiotic treatments to reduce its impact. Once a pen of fish is diagnosed with BKD, fish are considered chronically infected, potentially until harvest. Although there appears to be little or no evidence to support it, it is often assumed that subclinical infections affect productivity over the long term. We used a 2-stage hierarchical interrupted time series (ITS) analysis in an attempt to quantify the effect of subclinical BKD on mortality, growth, and food conversion ratio (FCR) of Atlantic salmon cultured in marine farms in Atlantic Canada. For all three outcomes, BKD had for some site cycles a positive effect, and for others a negative effect. Overall, the effect of BKD on mortality and growth could not be detected (effect -0.08 ((95% ci: -0.51, 0.35) and 0.00 (-0.02, 0.02)), while a very small effect showing an increase in FCR was detected (0.07 (-0.01, 0.15)). We hypothesized that minimal interference with fish performance may be compatible with the ecology of Renibacterium salmoninarum, the causative agent of BKD. For this organism, vertical transmission is a primary mode of propagation in low-density host populations as found in the wild. Since farms are always adapting and optimizing their farm management of BKD, these constant adjustments may also have negated our ability to detect the effect of many factors contributing to BKD productivity impacts. Hierarchical ITS analysis is considered an appropriate methodology to investigate the complex relationships with productivity measures over time under farming conditions. In the highly innovative salmon aquaculture industry, health records generating data available for time-series analysis is expected to become more accurate and abundant in the future, providing more opportunities for time-series regression studies.

Asunto(s)

Infecciones por Actinomycetales/veterinaria , Infecciones Asintomáticas/mortalidad , Enfermedades de los Peces/mortalidad , Análisis de Series de Tiempo Interrumpido , Enfermedades Renales/veterinaria , Micrococcaceae/fisiología , Salmo salar , Infecciones por Actinomycetales/microbiología , Infecciones por Actinomycetales/mortalidad , Animales , Acuicultura , Metabolismo Energético , Enfermedades de los Peces/microbiología , Enfermedades Renales/microbiología , Enfermedades Renales/mortalidad , Renibacterium , Salmo salar/metabolismo

RESUMEN

Renibacterium salmoninarum is the aetiological agent of bacterial kidney disease (BKD) in salmonid farms. This pathogen possesses at least three iron-acquisition mechanisms, but the link between these mechanisms and virulence is unclear. Therefore, this study used RT-qPCR to assess the effects of normal and iron-limited conditions on iron-uptake genes controlled by IdeR and related to iron acquisition in Chilean R. salmoninarum strain H-2 and the type strain DSM20767T . Further evaluated was the in vitro immune-related response of the Atlantic Salmon Kidney (ASK) cell line, derived from the primary organ affected by BKD. R. salmoninarum grown under iron-limited conditions overexpressed genes involved in haemin uptake and siderophore transport, with overexpression significantly higher in H-2 than DSM20767T . These overexpressed genes resulted in higher cytotoxicity and an increased immune response (i.e., TNF-α, IL-1ß, TLR1 and INF-γ) in the ASK cell line. This response was significantly higher against bacteria grown under iron-limited conditions, especially H-2. These observations indicate that iron-acquisition mechanisms are possibly highly related to the virulence and pathogenic capacity of R. salmoninarum. In conclusion, treatments that block iron-uptake mechanisms or siderophore synthesis are attractive therapeutic approaches for treating R. salmoninarum, which causes significant aquaculture losses.

Asunto(s)

Infecciones por Actinomycetales/veterinaria , Enfermedades de los Peces/inmunología , Hierro/metabolismo , Micrococcaceae/inmunología , Micrococcaceae/patogenicidad , Salmo salar , Infecciones por Actinomycetales/inmunología , Infecciones por Actinomycetales/metabolismo , Infecciones por Actinomycetales/microbiología , Animales , Línea Celular , Enfermedades de los Peces/metabolismo , Enfermedades de los Peces/microbiología , Inmunidad Innata , Micrococcaceae/metabolismo , Renibacterium , Virulencia

RESUMEN

PURPOSE: Membrane fluidity to a large extent is governed by the presence of branched-chain fatty acids (BCFAs). Branched-chain α-keto acid dehydrogenase (BKD) is the key enzyme in BCFA synthesis. A Staphylococcus aureus BKD-deficient strain still produced substantial levels of BCFAs. Pyruvate dehydrogenase (PDH) with structural similarity to BKD has been speculated to contribute to BCFAs in S. aureus. METHODOLOGY: This study was carried out using BKD-, PDH- and BKD : PDH-deficient derivatives of methicillin-resistant S. aureus strain JE2. Differences in growth kinetics were evaluated spectrophotometrically, membrane BCFAs using gas chromatography and membrane fluidity by fluorescence polarization. Carotenoid levels were estimated by measuring A465 of methanol extracts from 48 h cultures. MIC values were determined by broth microdilution.Results/Key findings. BCFAs made up 50 % of membrane fatty acids in wild-type but only 31 % in the BKD-deficient mutant. BCFA level was ~80 % in the PDH-deficient strain and 38 % in the BKD : PDH-deficient strain. BKD-deficient mutant showed decreased membrane fluidity, the PDH-deficient mutant showed increased membrane fluidity. The BKD- and PDH-deficient strains grew slower and the BKD : PDH-deficient strain grew slowest at 37 °C. However at 20 °C, the BKD- and BKD : PDH-deficient strains grew only a little followed by autolysis of these cells. The BKD-deficient strain produced higher levels of staphyloxanthin. The PDH-deficient and BKD : PDH-deficient strains produced very little staphyloxanthin. The BKD-deficient strain showed increased susceptibility to daptomycin. CONCLUSION: The BCFA composition of the cell membrane in S. aureus seems to significantly impact cell growth, membrane fluidity and resistance to daptomycin.

Asunto(s)

3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/metabolismo , Proteínas Bacterianas/metabolismo , Ácidos Grasos/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/enzimología , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida)/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/genética , Daptomicina/farmacología , Ácidos Grasos/química , Humanos , Fluidez de la Membrana/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo

RESUMEN

Infection with Renibacterium salmoninarum, the cause of Bacterial Kidney Disease (BKD) occurs in salmon populations in many locations, including the east coast of Canada. However, information about risk factors for BKD and their effects in the saltwater phase of the salmon aquaculture industry in the region is inadequate. We conducted a retrospective observational cohort study using industry health records in which BKD was recorded in New Brunswick, Canada, between 2006 and 2012. Several risk factors for BKD, such as stocking season, mortality percentage in the first four weeks, food conversion ratio (FCR), lice treatment, Bay Management Area (BMA), and production year were analyzed in a survival analysis using Cox proportional hazards models with cross-classified random effects to account for the structure of the data. The models incorporated effects on two different time scales, time since stocking and calendar time. The risk period was from stocking in salt water to first occurrence of clinical BKD in a pen. Results were time varying. Stocking season had a pronounced effect on time to clinical BKD after middle October of the first year after stocking, with clinical cases occurring less frequently in fall/winter-stocked fish compared to summer and spring-stocked fish; for example, in middle October, the Hazard Ratio of spring- compared to fall/winter-stocked fish was 15.8 (95% CI; 1.05, 354). Differences lasted until June and July of the second year after stocking. Effects of final hatchery before transfer to seawater, and egg source were not detected, but a limitation of this study was that this information was not available for 44.3% of the fish groups in our dataset. BKD status of a site/pen before fallow period and distance to nearest site with BKD were also not detected. Feed conversion ratio and mortality during the first four weeks affected BKD, indicating that better performing fish have a reduced hazard for BKD or vice versa, and implying that good general husbandry practices and BKD are correlated.

Asunto(s)

Infecciones por Actinomycetales/veterinaria , Enfermedades de los Peces/epidemiología , Enfermedades Renales/veterinaria , Micrococcaceae/fisiología , Salmo salar , Infecciones por Actinomycetales/epidemiología , Infecciones por Actinomycetales/microbiología , Animales , Acuicultura , Estudios de Cohortes , Enfermedades de los Peces/microbiología , Enfermedades Renales/epidemiología , Enfermedades Renales/microbiología , Nuevo Brunswick/epidemiología , Estudios Retrospectivos , Factores de Riesgo

RESUMEN

Branched-chain fatty acids (BCFAs) are important precursors for the production of advanced biofuels with improved cold-flow properties. Previous efforts in engineering type II fatty acid synthase (FAS) for BCFA production suffered from low titers and/or the co-production of a large amount of straight-chain fatty acids (SCFAs), making it nearly impossible for further conversion of BCFAs to branched biofuels. Synthesis of both SCFAs and BCFAs requires FabH, the only ß-ketoacyl-(acyl-carrier-protein) synthase in Escherichia coli that catalyzes the initial condensation reaction between malonyl-ACP and a short-chain acyl-CoA. In this study, we demonstrated that replacement of the acetyl-CoA-specific E. coli FabH with a branched-chain-acyl-CoA-specific FabH directed the flux to the synthesis of BCFAs, resulting in a significant enhancement in BCFA titer compared to a strain containing both acetyl-CoA- and branched-chain-acyl-CoA-specific FabHs. We further demonstrated that the composition of BCFAs can be tuned by engineering the upstream pathway to control the supply of different branched-chain acyl-CoAs, leading to the production either even-chain-iso-, odd-chain-iso-, or odd-chain-anteiso-BCFAs separately. Overall, the top-performing strain from this study produced BCFAs at 126 mg/L, comprising 52% of the total free fatty acids.

Asunto(s)

Acetiltransferasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Ácidos Grasos/biosíntesis , Ingeniería Metabólica/métodos , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa , Acetiltransferasas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Acido Graso Sintasa Tipo II/genética , Acido Graso Sintasa Tipo II/metabolismo , Redes y Vías Metabólicas/genética

RESUMEN

Two waxy rice (TNW1 and TCSW1, exhibiting high and low amylase activity, respectively), were stored at 4 and 17 °C (polished rice) and at room temperature (paddy rice) for 15 months. The fine structure of starch isolated from the aged rice and the pasting properties of starch and rice flour were studied. After storage, the percentage of short amylopectin (AP) chains increased in TNW1, and no uniform changing pattern was observed in the chain-length (CL) distribution of TCSW1. The viscosity of starch isolated from the aged rice increased as the storage temperature and duration increased. We hypothesised that this increase was due to the hydrolysis of AP by endogenous amylase and the generation of small clusters during storage, which caused the simple dissociation of AP and a high swelling degree of starch granules during gelatinisation. Factor analysis of the first two factors associated with the characteristics of viscograms and the CL of AP explained 72% of the total variation.

Asunto(s)

Oryza/química , Almidón/química , Harina/análisis , Almacenamiento de Alimentos , Estructura Molecular , Temperatura , Viscosidad

RESUMEN

During infection, macrophage lineage cells eliminate infiltrating pathogens through a battery of antimicrobial responses, where the efficacy of these innate immune responses is pivotal to immunological outcomes. Not surprisingly, many intracellular pathogens have evolved mechanisms to overcome macrophage defenses, using these immune cells as residences and dissemination strategies. With pathogenic infections causing increasing detriments to both aquacultural and wild fish populations, it is imperative to garner greater understanding of fish phagocyte antimicrobial responses and the mechanisms by which aquatic pathogens are able to overcome these teleost macrophage barriers. Insights into the regulation of macrophage immunity of bony fish species will lend to the development of more effective aquacultural prophylaxis as well as broadening our understanding of the evolution of these immune processes. Accordingly, this review focuses on recent advances in the understanding of teleost macrophage antimicrobial responses and the strategies by which intracellular fish pathogens are able to avoid being killed by phagocytes, with a focus on Mycobacterium marinum.

Asunto(s)

Infecciones Bacterianas/inmunología , Peces/inmunología , Fagocitos/inmunología , Animales , Proteínas de Transporte de Catión/metabolismo , Evasión Inmune , Inmunidad Innata , Espacio Intracelular , Óxido Nítrico/metabolismo